Unexpected Heme Redox Potential Values Implicate an Uphill Step in Cytochrome b6f

Cytochromes bc, key enzymes of respiration and photosynthesis, contain a highly conserved two-heme motif supporting cross-membrane electron transport (ET) that connects the two catalytic quinone-binding sites (Qn and Qp). Typically, this ET occurs from the low- to high-potential heme b, but in photosynthetic cytochrome b6f, the redox midpoint potentials (Ems) of these hemes remain uncertain. Our systematic redox titration analysis based on three independent and comprehensive low-temperature spectroscopies (continuous wave and pulse electron paramagnetic resonance (EPR) and optical spectroscopies) allowed for unambiguous assignment of spectral components of hemes in cytochrome b6f and revealed that Em of heme bn is unexpectedly low. Consequently, the cross-membrane ET occurs from the high- to low-potential heme introducing an uphill step in the energy landscape for the catalytic reaction. This slows down the ET through a low-potential chain, which can influence the mechanisms of reactions taking place at both Qp and Qn sites and modulate the efficiency of cyclic and linear ET in photosynthesis.


Detailed protocol of cytb6f isolation
Dimeric cytb6f complex was isolated from market spinach (S. oleracea) leaves using a large-scale protocol which was adapted from Baniulis et al. 1 and Romanowska 2 . Spinach leaves were homogenized in buffer 1 (50 mM Tris pH 8, 50 mM NaCl, 200 mM sucrose, 0.2 ml/L antifoam A) using a whole slow juicer. The resulting solution was filtered through a sieve and a must filter. After filtering, the solution was centrifuged at 17 000 g for 20 min, 4°C. The pellet containing chloroplasts was suspended in buffer 2 (10 mM Tris pH 7.5, 10 mM NaCl). Protease inhibitors (benzamidine, ε-amino caproic acid, and PMSF) were added to the chloroplast suspension before it was passed through a French Press (16 000 PSI). The resulting solution was centrifuged at 1000 g for 5 min, 4°C and the supernatant was ultracentrifuged at 148 000 g for 30 min, 4°C. The pellet was resuspended in buffer 3 (30 mM Tris pH 7.5, 50 mM NaCl, 1mM EDTA). The chlorophyll content in the suspension was determined using the protocol described by Porra, R.J. et al. 3 . The volume of the suspension was adjusted to a concentration of 1.5 mg/ml of chlorophyll by dilution with buffer 3. An equal volume of buffer 4 (buffer 3 supplemented with 52 mM octyl glucoside (OG), 0.2 % sodium cholate, and 400 mM ammonium sulfate (AS)) was slowly added to achieve selective solubilization of thylakoid membranes. The solution was subsequently stirred for 15 min and ultracentrifuged at 148 000 g for 20 min, 4°C. The supernatant was brought to 37% of AS saturation and centrifuged at 24 000 g for 10 min, 4°C. Then the supernatant was filtered through a 220 nm membrane and loaded onto a propyl-sepharose column preequilibrated with buffer 5 (buffer 3 with 1 mM undecyl α-D-maltoside (UDM) and 37% of AS saturation). The column was washed thoroughly with buffer 5 until the eluent was colorless. A greenish-brown band containing cytb6f was eluted with buffer 6 (buffer 3 with 1 mM UDM and 20% of AS saturation). The eluent was again brought to 37% of AS saturation and centrifuged at 24 000 g for 10 min, 4°C. After filtering through a 220 nm membrane, the supernatant was loaded onto a second propyl-sepharose column preequilibrated with buffer 5. The column was washed stepwise with buffer 5 containing decreasing amounts of AS (saturation decreasing from 35% to 30%), before a brownish band was eluted with buffer 6. The eluate was concentrated using microconcentrators (Merck Millipore) and the buffer was exchanged to buffer 7 (buffer 3 with 1 mM UDM). The sample was loaded onto a 10-25% continuous sucrose gradient in buffer 7 and ultracentrifuged at 141 000 g for 16 hours, 4°C. A brown band in the middle of the gradient containing pure cytb6f was carefully collected and the buffer was exchanged to buffer 7. Further biochemical analysis (including enzymatic activity, optical and EPR spectroscopy, electrophoretic analysis) indicated that this fraction contained a full complement of the cytb6f subunits and was enzymatically active.

Concentration and activity measurements
Concentration of cytb6f was determined spectrophotometrically by measurement of ascorbate-reduced minus ferricyanide-oxidized absorption spectra at 554 nm, relative to isosbestic point 543 nm, using differential extinction coefficient of heme f ε554-543≈ 25 mM -1 cm -1 4 . The enzymatic activity of cytb6f was determined by measuring the cytb6f-mediated reduction of plastocyanin. Preparation of substrates for activity measurement involved several procedures. Plastocyanin was oxidized with potassium ferricyanide which was subsequently removed by concentration-dilution cycle on microconcentrators. Decylplastoquinone was dissolved in ethanol and reduced to decylplastoquinol (dPQH2) with hydrogen gas in the presence of platinum on carbon as the catalyst. After reduction, dPHQ2 was diluted 3 times with dimethyl sulfoxide. The enzymatic reaction was carried out in buffer 3 (from isolation protocol) containing 20 μM of oxidized PC and 100 μM dPQH2. The total volume of the reaction mixture was 1 mL. The enzymatic reaction was initiated by injection of cytb6f to final concentration of 20 nM (concentration calculated per cytf) and the reaction progress was monitored using a Biologic Diode Array spectrophotometer. The reduction rate of plastocyanin was determined from the initial slope after the addition of cytb6f to the cuvette. The turnover rate of the sample was estimated to be approximately 120/s. Concentration and enzymatic activity of cytbc1 was assessed as described in 5 .

Statistical analysis
Figures S1 and S2 show the statistical analysis of equilibrium redox titration data obtained for cytb6f and cytbc1. As it is not possible to precisely assess the uncertainty of each of the external redox potential (Eh) value, it was assumed that the uncertainty is the same for all data points. The maximum value of uncertainty was assumed to be ± 15 mV, which was converted to standard uncertainty (u(Eh) = 8.66 mV) and used to create horizontal error bars. Figure S1 shows the analysis of the results obtained by decomposition of optical spectra. Due to the fact that standard errors of the amplitudes of reduced hemes were negligible, the points are indicated only as horizontal error bars. Figure S2 shows the analysis of the results obtained by analysis CW EPR spectra. Vertical error bars represent the uncertainties in determination of the amplitudes of the oxidized hemes. The uncertainty was calculated as standard uncertainty arising from both: the normalization of spectra on the amplitude of 2Fe2S and the noise present in the spectra.
The statistical significance of the model with respect to data was assessed by constructing confidence intervals at confidence level of 95%. Confidence intervals are depicted as blue confidence bands. Values of confidence intervals at each data point were calculated using the expression where: Ŷi is the value of fitted function at given Eh (Xi), t is the 100*(1-α/2) percentage point of Student's t distribution on n-p degrees of freedom (n = number of data points, p = number of parameters), RMSE is the Root Mean Squared Error, Xm is the mean of the Eh values and Sxx = Σ(Xi -Xm) 2 . Figure S1. Statistical analysis of the results, shown in Figure 2 in Main Text, obtained by decomposition of the optical spectra. a) and b) show the Nernst curves fitted to the reduced fractions of hemes bp and bn of cytb6f, respectively. c) and d) show the same type of analysis for hemes bl and bh of cytbc1, respectively. Data points are represented only by horizontal error bars for clarity. Data was fitted with the least squares method (Levenberg-Marquardt) using the Nernst function. Fitted values are given with the standard error calculated from the diagonal elements of covariance matrix. Confidence intervals at 95% confidence level are shown as blue bands.

Analysis of optical spectra
Low-temperature optical spectra of cytb6f and cytbc1 were analyzed using GAF procedure. The analysis was performed with a self-written Python program utilizing SciPy, NumPy and Pandas packages [6][7][8] . Each spectrum was fitted with a combination of three components: baseline, two-gaussian function and one-gaussian function. Baseline consisted of a spectrum recorded at high Eh where only hf and hc1 are reduced and a linear function to correct for slope deviation. Two-gaussian component was used to fit hbp and hbl while one-gaussian component fit hbn and hbh. Other variations of components including any combination of two or more one-or twogaussian functions were also examined but the best fit was obtained by fitting one two-gaussian component (hbp and hbl) and one one-gaussian component (hbn and hbh).
Implementation of GAF procedure was achieved by dividing the parameters into two sets: global, that should be held the same for all spectra and local, which could vary from spectrum to spectrum. The set of local parameters consisted of the amplitudes of the components and linear function arguments. The peaks widths, positions and relative amplitude (hbp and hbl ) were fitted as global parameters, since those values determined the shape of the component and should be the same for all spectra. Model consisting of 3 components with both global and local parameters was fitted to the data matrix using Levenberg-Marquardt algorithm. Reasonable boundary conditions were applied in the fitting procedure. The model fit the data very well as depicted in Figure 1 (Main text). Components for hemes b of both cytb6f and cytbc1 with optimized values are shown in Table 1 (Main text).

Analysis of EPR spectra
While EPR spectra of cytbc1 has been extensively studied and the EPR transitions of LS hemes b have been recognized, the presence of HS hcn in cytb6f creates additional EPR transitions originating from spin-spin exchange interactions with hbn. Typical CW EPR spectra of cytb6f measured at 3 different Eh are shown in Figure S3. A rhombic EPR spectrum at 1.76 < g < 2.02 originates from the reduced 2Fe2S cluster. The signal of hf (g = 3.51) was not detected, as this heme was fully reduced. The transition at g = 3.67 was previously ascribed to gz transition of the oxidized, highly axial low-spin (HALS) hbp. The signals at g > 4.3 were ascribed to hcn which is spin-coupled to hbn. An additional component at g ~ 6 of unknown species, suggests a presence of a HS heme of axial symmetry. It is likely that it originates from either partially denatured hf or a heme b, or from a small fraction of hcn which is not coupled to hbn. Similar axial HS signal of heme was detected in cytochrome c oxidase, despite the presence of spin-spin exchange interaction with the copper ion.
Well separated EPR signals ascribed to the pair of hcn and hbn were found at g = 12.4 and 4.73, thus they were used as a measure of the redox-dependent changes in the amplitude of oxidized hcn to construct the Nernst curves for this particular pair of hemes.

Global analysis fit of ESE decay curves
The model used for the global analysis fit of ESE decay curves included following assumptions: a) only dipolar interactions within a single monomer were considered; b) a number of fast relaxing species having different Em in cytb6f was 3 -hemes bn, bp and cn; c) dipolar ESE decay curve resulting from the interactions between 2Fe2S and a heme iron was approximated with monoexponential function described by average Tdip; d) effect of changes in the redox state of heme cn and bn on ESE decay of 2Fe2S were treated separately, despite existence of spin-spin exchange interactions between these two hemes; e) for cytbc1 the number of species was 2, while the second species representing heme bh was divided into two fractions with the lower and higher Em and contributions of 60% and 40%, respectively.
The first and the second assumptions were supported by the observation, that increasing the number of species from 3 to 4 did not lead to any sensible results of the fit and the obtained parameters of the redundant 4 th species (Tdip, fraction and Em) were always very similar to one of the remaining species with shared contribution to compensate the respective fractions. A model used for fitting to the whole experimental set of ESE decay curves is described by the following formula: eq. S2 where: of the phase relaxation of 2Fe2S; Tdip1, Tdip2 and Tdip3 are the semi-quantitative time constants associated with the enhancement of the phase relaxation imposed on 2Fe2S by a respective fast relaxing species; t and Eh are independent variables representing the time axis of a single ESE decay curve and the external redox potential, respectively. The fractions f1, f2 and f3 (fx) were calculated on the basis of the Nernst equation, as a function of Em, n and Eh for a particular sample: eq. S3 where: fx -fraction of species x, Emx -the redox midpoint potential of species x. For the reasons given in the main text the parameter n was fixed at 1.
By combining eqs. S2 and S3, we obtained the model, in which all experimental ESE decay curves, at different Eh, were described by 9 global, free adjustable parameters: 3 Em, 3 n and 3 Tdip values.

Measurement of Eh-dependent enhancement of the phase relaxation of 2Fe2S in cytb6f.
Global fitting of the model to ESE decay curves of cytb6f with all 9 parameters to optimize was found to be unstable resulting mostly in unreliable values of n, which entails random combinations of Em and Tdip. Fixing n values at 1 greatly improved the stability of the optimization. The convergence criteria was tested by performing 1000 independent optimizations, starting from randomized set of initial guesses for Tdip, Em, and fixed n =1. Values of Tdip were randomly drawn from the range 0.1 to 20 μs, while Em values were randomly drawn from the range -200 to +200 mV. Among 1000 optimizations 573 were successful, while 424 did not reached the global minimum, showing that a probability of successful optimization was 0.573 +/-0.016. All successful optimizations yielded nearly the same values of Tdip and Em for respective species. The results of the fitting was shown in Table 3 in the Main Text.
We also performed another fit with parameters of Em and n fixed at values determined by optical and CW EPR spectroscopy (Figures 2 and 3): Em = -111 mV and n=1 (species 1 -heme bn); Em = -80 mV and n = 0.7 (species 2 -heme bp), Em = +46 mV and n = 1, (species 3 -heme cn). In this case, the only adjustable free parameters were Tdip.
In both these cases Tdip values are the lowest for the species 1 with the lowest Em suggesting, that it is more remote from the 2Fe2S cluster than the species with intermediate Em value.

Measurement of Eh-dependent enhancement of the phase relaxation of 2Fe2S in cytbc1.
We compared the results of measurement of ESE decay in cytb6f to the data obtained in similar manner for cytbc1. In this case the model was slightly modified to account for the presence of two fractions of heme bh with higher (bhh) and lower (bhl) Em. These two fractions of the heme bh were expected to have the same dipolar effects on the phase relaxation of 2Fe2S as they correspond to the same structure, distance and paramagnetic properties. The fit with fixed Em and n parameters (drawn from optical spectroscopy data) yielded Tdip of heme bl less than that of heme bh which could be expected as both these hemes possess similar paramagnetic properties and the difference in dipolar effect on phase relaxation of 2Fe2S results from heme bl being closer to 2Fe2S than heme bh.
The result of fitting of the above discussed models to ESE decay curves of cytb6f and cytbc1 are presented in Figure S4. = 10 0.01666⋅ ⋅( ℎ − ) 1 + 10 0.01666⋅ ( ℎ − ) Figure S4. Results of application of GAF to ESE DC curves obtained for cytbc1 and cytb6f. a) the concatenated ESE DCs measured for cytb6f at different Eh (blue) and the GAF curves (red). b) Difference between ESE DCs and the fitted model. Panels c) and d) represent the same analysis as for a) and b), respectively but obtained for cytochome bc1. The x-axes represent consecutive points of ESE DCs, that were concatenated starting from the lowest (left) to the highest Eh (right). Before analysis each ESE DC was normalized by division of ESE DC amplitude by estimated amplitude at the zero time point (the onset of the relaxation process). Initial 440 ns of the ESE DC are missing as being obscured by the resonator dead time.
Overall, the analysis of ESE decay curves measured for cytb6f provided estimates of Ems for the 3 species but gave no explicit information on which heme was represented by the respective species. However, comparison of Em values obtained by the analysis of relaxation curves with those obtained from optical and CW EPR spectroscopy (see Figures 2 and 3 in the Main Text) suggests that Em of species 1 is similar to Em of heme bn (species having Em around -129 mV (Figure 2b)) and therefore it is most probably heme bn. Species 2, with Em = -96 mV has similar Em value to heme bp (Figure 2a). Both these comparisons correspond well with the spatial arrangement of hemes in the structure of cytb6f given that the Tdip obtained for species 1 is lower than that of species 2 and heme bn is placed more remotely from 2Fe2S than heme bp. Species 3 was optimized with Em value around + 20 mV which is close to the value obtained for heme cn (Figure 3b).

Discussion on relaxation enhancement
We performed analysis of pulse EPR data of cytb6f and the results were compared to the data obtained for structurally similar protein cytbc1. Figure 4 in the Main Text compares the distances between paramagnetic 2Fe2S center and heme iron ions in both cytochromes. In the case of cytbc1, heme bl is positioned closer to 2Fe2S (approx. 26.4 Å) than heme bh (approx. 34.2 Å), therefore assuming that these two hemes have similar paramagnetic properties the former heme should exert a stronger dipolar effect on the phase relaxation of the 2Fe2S cluster than the latter. Therefore, a shorter Tdip for heme bl-2Fe2S interactions was expected. Similar effect was expected for cytb6f in which heme bp is also closer to the 2Fe2S cluster than heme bn (~ 30.5 Å and ~39.4 Å). Theoretically, dipolar relaxation curves are not mono-exponential as they depend on many additional factors such as relative angles between the vectors connecting the paramagnetic centers and the external magnetic field. However, we performed a qualitative comparison of the relative dipolar relaxation time constants by taking into account only relative distances and Tdip values of cytb6f and cytbc1. In the most simplified case, the strength of the dipolar effect on the phase relaxation can be described by a formula which considers distance r between two interacting centers: where: <Tdip -1 > is the average, effective dipolar ESE decay rate caused by the dipolar interactions, <a> is the proportionality factor including all remaining effective contributions other than distance averaged, while r is the distance between 2Fe2S cluster and respective heme iron ion. First we calculated average <a> values for interactions between hemes bp, bn, cn and 2Fe2S in cytb6f and corresponding <a> for interactions between heme bl and bh and the 2Fe2S cluster in cytbc1 with the use of Tdip values obtained for the respective cytochromes. In case of cytb6f, the averaged <a> value was estimated to ~27.5 Å 3 ×GHz, while for cytbc1 this value was similar and equal to ~30.0 Å 3 ×GHz. Using these different <a> values for calculating distances between 2Fe2S cluster and hemes in both cytochromes and respective Tdip values, we obtained distances ~34.4 or ~36.8 Å for 2Fe2S -heme bp, ~38.6 or ~39.4 Å for 2FeS2 -heme bn and ~44.0 or ~41.7 Å for 2Fe2S -heme cn. In cytbc1 the estimated distances were ~28.5 and ~32.1 Å for 2Fe2Sheme bl and 2Fe2S -heme bh, respectively. These values however must be treated only as crude approximations since such an analysis ignores many factors that contribute to the effective enhancement of the phase relaxation of the cluster. Most of the additional factors are not available and, additionally, we must take into account the fact that the PDB structures do not provide information on the distribution of the position of 2Fe2S due to the mobility of the iron-sulfur protein, an inherent feature of cytbc1 and cytb6f. However, despite the above-mentioned limitations in the analysis of the pulse EPR data, a general view drawn from these experiments is qualitatively in agreement with the expected larger effects of hemes bp/bl on the relaxation of 2Fe2S than hemes bn/bh due to the differences in their relative distances to the cluster and their estimated Em values. This again supports the thesis that heme bp in cytb6f exhibits a higher Em than heme bn, whereas in cytbc1 the respective heme bl and bh are low-and high potential LS heme.